Epidemiological evidence suggests that asthma has its origins in early life. It is speculated that maternal exposures to environmental factors affect the immunological sensitization and lung growth of the neonate through epigenetic modifications of the fetal gene transcription; which determines the offspring susceptibility to allergic airway hyperresponsiveness. DNA methylation, the most-studied epigenetic mechanism, has been shown to be affected by prenatal or postnatal exposure to allergens, diesel exhaust and cigarette smoke, and these have been linked to increased asthma risk. However, few studies to date have explored (1) the in utero allergen exposure window that results in later sensitivity to asthma in the offspring and (2) the aberrant DNA methylation changes induced by maternal allergen exposures that can persist in subgenerations. To address these gaps in the current knowledge, we have used a mouse experimental asthma model to examine epigenetic changes in successive generations of offspring whose mothers/grandmothers were exposed to house allergens. Offspring of mothers who exposed to house-dust-mite (HOM) two weeks before conception to embryonic day 7, showed decreased airway hyperresponsiveness (AHR) while the offspring of mother who exposed to HOM two weeks before conception to weaning, had increased AHR as compared to offspring of unexposed mother. The changes in the offspring's AHR phenotypes were associated with lung lfnv and Acs/3 DNA methylation. Strikingly, we further found that maternal exposure to allergens impacts the inheritance of AHR phenotypes in the third successive generation (F3). Hypothesis: Based on these preliminary findings, we hypothesize that maternal exposure to HOM alters the epigenome of the fetus and modulates the lung growth in the neonate. The timing of these prenatal and neonatal exposures is critically important in determining both epigenetic and pathologic phenotypes that develop in the lung of the progeny and persist in subgenerations. Specific Aims: To test our hypothesis, we proposed two complementary aims: 1) define the critical time window for epigenetic reprogramming of lung development and its association with asthma risk later in life; 2) investigate if germline epimutations transmit to subgenerations (in somatic cells) and contribute to allergen induced AHR. Innovation: We will utilize advanced single-cell bisulfite deep-sequencing and novel computational analysis to identify epigenetic changes underlying the fetal origins of asthma diseases. Significances: To summarize, in this R21, we plan to identify the links between maternal allergens exposure, DNA methylation changes, embryonic lung physiological changes and asthma disease susceptibility. Defining the critical exposure window that leads to these pathologic epigenetic effects could ultimately be important for pregnant women living in allergenic environments. Our findings will provide insights for improving early intervention strategy to reduce the asthma risk; given the fact that aberrant DNA methylation can be reversible.